Abstract Children diagnosed with Diffuse Intrinsic Pontine Gliomas (DIPGs) are faced with a mortality rate of 100%. The current standard of care is radiation therapy. Despite achieving initial responses, tumors quickly exhibit resistance and start to grow again. We have taken leading positions in a national trial, DIPG-BATs, that has evaluated routine biopsy of DIPGs in children. We seek to take advantage of the tissue obtained through the DIPG-BATs trial to understand the genetic underpinnings of DIPG and their impact on therapeutic response. We also seek to identify therapeutic combinations that are sufficient to prevent the acquisition of radiation resistance. In Aim 1, we propose to analyze the largest set of DIPG whole genomes to date. We will combine sequencing data collected on the DIPG-BATs trial with those from newly diagnosed patients, in addition to previously published genomes. In Aim 2, we will evaluate the role of PPM1D mutations in generating resistance to radiation therapy. Our initial analysis of BAT biopsies has revealed that over 50% of DIPG genomes contain mutations in either PPM1D or TP53, and that these mutations are mutually exclusive. PPM1D has been characterized as a negative regulator of TP53, a critical facilitator of radiation sensitivity. In Aim 3, we will use both hypothesis- based and unbiased approaches to identify therapeutic combinations with radiation that increase efficacy. We will utilize genetic and pharmacological methods of inhibiting PPM1D and MDM2 in combination with radiation in patient-derived DIPG lines with TP53 mutations. We will also perform a genome-wide CRISPR-cas9 screen to identify genes whose suppression selectively increase radiation response in DIPG-relevant cell lines. These experiments will address at least three central questions regarding resistance to radiation therapy in DIPG: characterization of driver genomic alterations and identification of those that confer resistance to radiation therapy, determination of how alterations in p53 signaling confer resistance to radiation, and evaluation of PPM1D as a novel therapeutic target.